The present invention relates to novel recombinant nucleic acids and methods for their use to introduce a prokaryotic genome or other valuable DNA into a eukaryotic cell as a circular molecule that is then converted to a linear automatic eukaryotic artificial chromosome within the eukaryotic nucleus and as a circular chromosome within a membrane bound, extranuclear element. Such added prokaryotic genomes or other valuable DNAs should add new functions to the eukaryotes or allow them to be selected for using this hybrid system.
Evolutionary theory proposes that mitochondria and plastids originated by engulfment or cell fusion of prokaryotes by eukaryotes. As this relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome (Cavalier-Smith. (1987) Ann. NY Acad. Sci. 503:55-71). Support for this theory is found in the fungus, Geosiphon pyriforme, which contains in its hyphal system cyanobacteria belonging to the genus, Nostoc, but which retain the capacity for autonomous growth and replication (Mollenhauer. (1992) Geosiphon pyriforme, In Algae and Symbiosis. Biopress Ltd., Bistol, pp. 339-351). Additional support is found in algae which have plastids containing DNA that has a significant level of homology and similar gene organization to cyanobacteria but the plastids have lost most of the cyanobacterial genes to the cell nucleus (Douglas. (1994) Chloropast Origins and Evolution, In Molecular Biology of Cyanobacteria, vol. 1 (Bryant, ed.) Kluwer Academic Publishers, Boston, pp. 91-118). The reason and mechanism for the relocation of a large proportion (greater than 90%) of the bacterial genes to the nucleus are unknown (Valentin et al. (1992) Phylogenetic origin of the plastids, In Origins of Plastids (Lewin, ed.) Chapman and Hall, New York, pp. 193-221).
It would therefore be useful to develop a system to introduce an entire prokaryotic genome into a eukaryotic organism and to study the interactions of the two genomes and the effect this has on both organisms. Preferably, such a system would permit both nuclear and extra-nuclear localization of the bacterial genome. This system also would provide a model for the evolution of mitochondria, chloroplasts, and other plastids.
The present invention presents new technology that can be used to transfer entire bacterial chromosomes into yeast or other eukaryotic organisms in such a manner that they become functional linear artificial chromosomes and, furthermore, may become compartmentalized bacteria or organelle-like structures. The bacterial chromosome will be expressed partially in the nucleus and in the bacterial organelle and provides new and useful pathways to the eukaryote host immediately after formation of the hybrid cell or after selections for specific desired functions norm ally done by the prokaryote alone as well as new functions. These new vectors and methods additionally provide a means to efficiently introduce very large segments of DNA into eukaryotic cells without extracellular manipulation.
The present invention provides compositions and methods for transferring an entire prokaryotic genome or other DNAs into a eukaryotic organism. In one aspect, the invention provides a recombinant expression system to introduce an endonuclease gene with a rare cleavage site into a eukaryotic organism as well as using endonuclease(s) already present in the eukaryote. In another aspect, the invention provides circular recombinant nucleic acids that are converted by the endonuclease to automatic, eukaryotic artificial chromosomes. The invention also provides a recombinant nucleic acid for converting a prokaryotic genome into a eukaryotic chromosome. In yet another aspect, the invention provides methods for introducing converted bacterial genomes and circular DNAs into a eukaryotic organism. Finally, the invention also provides methods for selecting eukaryotic organisms comprising the modified bacterial chromosome or other DNAs, as well as the selectable addition of new valuable functions from the prokaryotes or other DNAs being added to the eukaryotic cell.